Project Summary Although designed to facilitate home testing, rapid diagnostic test (RDT) have typically been used in a hospital- based point-of-care (POC) setting for diagnosis of symptomatic patients where parasite density is high (>2000 per microliter of blood). This is, in part, due to the lack of sensitivity and stability of current lateral flow-based RDT devices. In the current project, we propose to develop a novel on-demand paper-based diagnostic platform that will enable self-testing (or field analysis) followed by remote and centralized signal detection using portable mass spectrometers. This diagnostic approach is innovative as it combines new levels of simplicity and practicality, modest levels of cost, and a centralized detection strategy, which will redefine the breadth of application and performance/cost ratio for accurate malaria detection. More importantly, our proposed approach will provide an opportunity to diagnose asymptomatic patients before the disease becomes clinically apparent, therefore providing affordable community-based surveillance and POC tests. We will achieve this objective through the rational design of novel ionic probes for coupling to specific antibodies, which will subsequently enable the implementation of a paper-based immunoassay platform utilizing vertical fluid flow format. Compared with enzymes, these stable ionic probes make possible the ability to interrupt, store, and restore the immunoassay test, allowing detection at a later convenient time. In addition, the ionic probes are rationally designed to yield ions of small masses upon stimulation permitting the use of portable instruments for field analysis. By targeting all three levels of malaria care (i.e., symptomatic POC patients, surveillance-based asymptomatic detection and field analysis in an outbreak), we will offer a greater chance of limiting the 188 million cases of malaria and reduce the $12 billion yearly loss in worker productivity. Aim 1 is to design and synthesize active (cleavable) ionic probes as mass reporters for our proposed mass spectrometry (MS)-based immunoassay technology. Two signal amplification strategies are also proposed to enable ultrasensitive detection of low parasite density from a finger prick blood. Aim 2 will develop and optimize the paper-based MS immunoassay platform by implementing two interrelated tasks: establishment of ionic probe- mediated immunoassay on ordinary paper substrate, and the development of new on-chip MS detection methods to facilitate the ease of use of the paper devices. Aim 3 involves validation of the method, through close collaborative interactions with two molecular biologists (Kingsley Badu ? not related to PI; KNUST, Ghana, and Dr. Cristian Koepfli - University of California, Irvine, Public Health). Dr. James Odei (Clinical Biostatistics, Ohio State University) is included for proper data analysis and interpretation. As part of this aim, we project screening of at least 1,600 patients/volunteers with Plasmodium (P) falciparum (in Ghana) using the developed paper device; we expect to include ~50% children under age 15. The method will also be validated against P. vivax malaria samples (~600 volunteers expected) collected from Ethiopia.